The broad, long-term objective of this application is to develop a practical, gene therapy-based protocol to induce effective, long-term tolerance to GAD, a primary autoantigen in diabetes, in the hope that this will lead to an effective therapy for this debilitating disease. One of the best hopes for developing effective therapies for autoimmunity is to establish protocols that induce antigen-specific tolerance to the primary initiating autoantigen. In this application, we will examine the potential of a novel method for inducing long-term tolerance to GAD in non-obese diabetic (NOD) mice. This application will test the following hypothesis: The targeted expression of a primary autoantigen in the endocytic compartment of hematopoietic cells will result in effective, long-term tolerance induction and prevention of autoimmunity. This hypothesis is based on the belief that poor tolerance induction to the primary autoantigen is often due to its sequestrated location (cellular and/or anatomical) and thus poor presentation of peptides by major histocompatibility complex (MHC) class molecules. If GAD were expressed as widely, and presented as efficiently, as common-antigens, such as MHC class I molecules, we believe that autoimmunity would be prevented. The following Aims will be pursued: Specific Aim 1: We will examine whether targeted expression of GAD can increase the efficiency of peptide presentation to GAD-specific T cells. A series of enzymatically inactive GAD mutants have been generated to target expression in specific cellular locations in order to optimize presentation by MHC class II molecules. This aim will verify the location of these mutants by confocal microscopy and evaluate their ability to stimulate GAD-specific T cells. Specific Aim 2: We will also examine whether the targeted expression of GAD in hematopoietic cells can result in effective, long-term tolerance induction in NOD mice. Retroviral-mediated stem cell gene transfer will be used to express the GAD mutants in all NOD hematopoietic cells. The toleragenic state and/or T helper (Th) phenotype of any detectable GAD-specific T cells will also be determined. The incidence of insulitis and diabetes will be monitored to determine the extent of tolerance induction and prevention of disease.